|Gadaleta, Agata - UNIV OF BARI, ITALY|
|Giancaspro, Angelica - UNIV OF BARI, ITALY|
|Blanco, Antonio - UNIV OF BARI, ITALY|
Submitted to: Journal of Cereal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 5, 2007
Publication Date: September 1, 2008
Citation: Gadaleta, A., Giancaspro, A., Blechl, A.E., Blanco, A. 2008. A Transgenic Durum Wheat Line that is Free of Marker Genes and Expresses 1dy10. Journal of Cereal Science. 48:439-445. Interpretive Summary: Current methods for genetic engineering of wheat require the use of selective agents, such as herbicides and antibiotics, and marker genes for resistance to identify the few percent of cells that receive the incoming DNA. In this paper, a variation of the biolistic method of transformation is used to obtain durum wheat transformants that contain no antibiotic or herbicide resistance genes. The transforming DNAs consisted of the minimum sequences needed for expression in a wheat plant: the promoter, coding sequence and transcription terminators. The selection marker gene allows wheat cells to grow in tissue culture on mannose as a sugar source. Two other genes were included, both encoding high-molecular-weight glutenin genes from wheat that are associated with good bread-making quality. Transformation efficiencies were the same as with the standard methods of wheat transformation. In one line, the marker and wheat glutenin genes integrated into separate chromosomal sites and sublines that had inherited only a wheat glutenin transgene were derived. The resulting wheat plant is transformed, but only contains wheat DNA sequences. The use of these methods may make wheat biotechnology more acceptable to consumers because no foreign DNA remains in the resultant plants.
Technical Abstract: We used a combination of “clean gene” technology and positive selection to generate transgenic durum wheat lines free of herbicide and antibiotic resistance marker genes. Biolistic transformation experiments were carried out using three “minimal gene cassettes” consisting of linear DNA fragments excised from the source plasmids. The targeted trait genes were two bread wheat sequences encoding the Dx5 and Dy10 high-molecular-weight glutenin subunits (HMW-GS), which have been associated with superior bread-making quality and which are absent from durum wheats. The positive selectable marker was the E. coli phosphomannose isomerase (pmi) gene, whose product allows plant cells to utilize mannose as a carbon source. PCR assays of genomic DNA identified 15 regenerated plants that contained the pmi marker gene for a transformation efficiency of 1.5%, which is similar to that of durum wheat with intact circular plasmids. Line TC-52 was investigated for four generations to study inheritance and expression stability of pmi, non-expressed 1Dx5, and expressed 1Dy10 HMW-GS transgenes. From these analyses, we observed that the 1Dy10, 1Dx5 and pmi transgenes were not linked, allowing us in the T3 generation to identify 1Dy10 transgenic segregants that contained no marker or silent 1Dx5 transgenes. These experiments show that it is possible to combine biolistic transformation by minimal gene cassettes with genetic segregation to make marker-free transgenic wheat plants with new traits.